G protein-coupled receptors (GPCRs) mediate hormonal control of numerous intracellular effectors. An important mechanism for controlling GPCR signaling involves stimulus-dependent phosphorylation of the receptor, a process primarily mediated by G protein-coupled receptor kinases (GRKs). GRK-mediated receptor phosphorylation promotes the binding of arrestins, which function in receptor/G protein uncoupling and subsequent GPCR endocytosis. GRKs are modular proteins consisting of an N-terminal RGS homology (RH) domain, a central protein kinase catalytic domain, and a C-terminal lipid-binding domain. In this application, we propose to continue our broad-based studies aimed at defining the role of GRKs in regulating cell signaling. Three specific objectives are proposed. 1. Elucidate the molecular mechanism of GPCR binding and activation of GRKs. While GRKs specifically bind to agonist-occupied GPCRs, little is known about the specific residues that mediate receptor binding or how such binding results in GRK activation. We propose two major lines of investigation to test the hypothesis, that GPCR binding is primarily mediated by the catalytic domain. The first will involve generating a series of point mutations based on the recently solved X-ray structure of GRK2. These mutants will be expressed, purified, and analyzed for their ability to bind and phosphorylate various receptor and non-receptor substrates. The second line of investigation will involve expressing and characterizing a catalytic domain construct of GRK2. Taken together these studies should enable us to better define the residues that mediate GRK interaction with GPCRs and elucidate the mechanisms involved in GRK activation. 2. Characterize the functional role of the GRK RH domain. While recent studies have demonstrated that the GRK RH domain interacts with Galphaq family members, the specificity and functional consequences of this interaction remain poorly defined. Interestingly, preliminary studies reveal that GRK2 and GRK3 specifically interact with Galphaq, alpha11, and alphal4 while GRK4 family members interact selectively with Galpha16. The molecular basis for these differences will be further elucidated by site directed mutagenesis and functional analysis of the various GRK RH domains. In addition, we will test the hypothesis that the GRK RH domain functions as an effector for Gaq family members and will test this by characterizing the effects of wild type and Galpha-binding defective GRKs on agonist-dependent phosphorylation and signaling of several GPCRs. 3. Characterize the biology of GRKs using C. elegans as a model organism. C. elegans has served as a powerful model to study a wide variety of biological processes. Interestingly, the C. elegan's genome encodes approximately 1200 GPCRs, 21Galpha subunits, and 12 RGS proteins but only 2 GRKs and a single arrestin. In an effort to better define the biological role of GRKs and correlate structural features with in vivo function, we propose to study the C. elegans GRKs. GRK biology will be studied using knockdown (RNAi and chemical mutagenesis) and transgenic (overexpression of wild type and mutant GRKs) approaches and subsequent analysis of basic biological processes such as egg laying, locomotion, chemotaxis,and adaptation. These studies should enable us to define the biological role of the individual GRKs in C. elegans and start to elucidate the role of specific protein interactions in GRK function. Overall, these studies should more clearly define the molecular and biological role of GRKs in regulating cell signaling.